The overall goal of this project has been to provide a biochemical mechanism for formation of selenite cataract in young rats. Our previous work has shown that partial proteolysis of Beta-crystallins by calcium- activated protease, calpain II, plays a central role in the mechanism of selenite cataract. The current proposal is focused on providing a biochemical mechanism on how partially truncated B-crystallin polypeptides become insoluble in selenite cataract and other models of cataract using older rat lenses. Aim #1 will use an in vitro model of crystallin insolubilization to determine why removal of the N-termini on B-crystallin subunits by calpain causes insolubilization. Aim #2 will directly compare results of the in vitro studies of proteolytic insolubilization to insolubilization occurring in the in vivo model of selenite cataract and in other models of cataract using older rat lenses. Aim #3 will determine the role of a-crystallin chaperon activity in proteolytic insolubilization of B-crystallins. Techniques to be used include: two dimensional electrophoresis followed by N-terminal protein sequencing of spots, fast atom mass spectroscopy, laser light scattering, UV circular dichroism, protein-protein cross- linking, and turbidity measurements. The results will extend our on- going studies concerning how crystallins become insoluble in cataracts and how crystallins interact in normal lens. This knowledge may be used in the future to develop drugs for prevention of cataract by maintaining the solubility of lens proteins.